Pulmonary fibrosis has emerged as the leading cause of death in patients with systemic sclerosis (SSc)/scleroderma, yet currently available therapies are only marginally effective. While recent work from several groups of investigators suggests a key role for lung macrophages in the development of pulmonary fibrosis in patients with SSc, this remains an area of controversy as the specific population(s) of lung macrophages that drive fibrosis and the molecular mechanisms by which they do so remain largely unknown. In advance of this proposal, we have developed novel tools to elucidate macrophage heterogeneity in the mouse and human lung including lineage tagging, flow cytometry and transcriptional profiling in order to examine the differential role of tissue-resident alveolar macrophages, as compared to the monocyte-derived alveolar macrophages recruited to the lung, in the pathogenesis of fibrosis. Using unbiased transcriptional analysis (RNAseq) of flow-sorted macrophage populations over the course of the development of experimental fibrosis, we identified genetic signatures of monocyte to alveolar macrophage differentiation and fibrosis. Moreover, we have developed a murine model, which lacks the pathogenic monocyte-derived macrophage and is thus unable to develop lung fibrosis. Specifically, mice with macrophage-specific deletion of caspase-8, a cysteine-aspartic acid protease originally identified as a key initiator of the apoptotic death receptor pathway and suppressor of necroptosis (CreLysMCasp8fl/fl or CreCD11cCasp8fl/fl), showed significantly attenuated fibrosis and an inability for recruited monocytes to differentiate into pro-fibrotic monocyte-derived Siglec Flow macrophages following intratracheal treatment with either bleomycin or an adenovirus encoding an active form of TGF-? as compared to Casp8fl/fl, CreLysM or CreCD11c mice. Thus, this model is an ideal to tool to understand the differentiation of monocytes into pro-fibrotic alveolar macrophages in response to lung injury. These model systems will allow us to apply the transcriptional data to the study of monocytes from patients with SSc differentiated into alveolar macrophages in the lungs of humanized mice and alveolar macrophages obtained from patients with SSc at the time of lung transplantation. Our studies will test the hypothesis that monocyte differentiation into pro-fibrotic Siglec Flow alveolar macrophages is essential for the development of lung fibrosis in both mice and humans in three interrelated specific aims.